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Inaugural dissertation--Berlin. / At head of title: Aus der Frauenklinik der Kgl. Charité zu Berlin ... Lebenslauf. "Literatur": p. [45]-46.

Thesis (M.S)--University of Michigan, 1975. / Typescript (photocopy). Includes bibliographical references (leaves 46-50). Also issued in print.

Thesis (M.S)--University of Michigan, 1975. / Typescript (photocopy). eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 46-50).

Thesis--Leyden. / Summary in Dutch. eContent provider-neutral record in process. Description based on print version record. Includes bibliographies.

The main aims of this work were to develop and test methods for the analysis of the shape and calcium binding properties of fibrinogen and its degradation product fragment D. The techniques of photo-sensitized labelling and cross-linking were used to obtain information about the shape of these proteins and both revealed interesting information. Photosensitized labelling proved particularly useful and results obtained using this technique suggested that fragment D (Ca++) in solution and the fragment D domains of fibrinogen are conformationally very similar. These studies also suggested that the A chain of fibrinogen is highly exposed at the surface of the molecule and that it shields other portions, particularly the D-domains of the molecule. The use of chemical cross-linking reagents revealed a gross-conformational difference between fragment D (Ca++) and fragment D (EDTA) and it was further shown that this difference is a result of cleavage of the chain of fragment D (Ca++) rather than calcium removal. This suggests that the calcium ion bound by fragment D (Ca++) exercises a protective influence over a plasmin susceptible bond and that cleavage of this bond allows a gross conformational change. This is consistent with the results from photo-sensitized labelling which showed the C-terminal portion of the chain of fragment D to the surface exposed. It was also shown that the technique of photo-sensitized labelling could be modified to induce cross-linking yielding a plasmin digestable product. These studies confirmed results from labelling studies which suggested that the chain C-terminii protect the N-terminal portion of fibrinogen. Calcium binding studies revealed two classes of high affinity calcium binding site in fibrinogen. Two sites of high (Kd > 10-5m) were shown to exist in the D-domains of the molecule, a third site of even higher affinity (Kd > 10-7m) was also found although the position of this site was not identified. These results of the study may be used to reconcile some of the more extreme views of fibrinogen shape as they suggest that fibrinogen is a protected trinodular structure.

There is much controversy regarding the conformation of fibrinogen. Several models have been proposed ranging from a trinodular arrangement to a globular conformation. It has also been suggested that fibrinogen has a flexible structure where the shape of the molecule is influenced by its environment, one major factor being calcium concentration, In addition, although the importance of tightly bound calcium ions (kd 1M) to the fibrinogen molecule is well established, the role of the larger number of low affinity sites (kd 1mM) is still a matter of some debate. In this study, the two techniques of photosensitized radioactive surface labelling and photosensitized cross-linking were selected for development and assessment. This was done with a view to examining the conformation of fibrinogen in its native state and under different solvent conditions, with particular reference to the influence of calcium. The two techniques have been shown to have definite applications in their use as probes into the conformation of fibrinogen. Results derived using these methods support the view put forward by various authors that fibrinogen is a dynamic molecule having a flexible conformation and that the conformation adopted is dependent on solvent composition. Calcium concentration in the millimolar range is particularly significant and consequently so is the saturation of the low affinity binding sites which may well have a regulatory function. Experimentally two extreme conformations have been demonstrated, - a closed, compact structure at low calcium concentrations compared to a more open one at higher calcium concentrations. More importantly, the techniques used also show the subtle changes which occur within the molecule as the calcium concentration is raised, changes which may be more significant physiologically. The most important of these are the effect of calcium on the C-terminal parts of the Aa-chains and the D domains of the molecule.